Phenotypic bacterial epidemiology and antimicrobial resistance profiles in neonatal sepsis at Jimma medical center, Ethiopia: Insights from prospective study

Background Epidemiological profiles and the rundown crisis of antimicrobial resistance from bacterial isolates in neonatal sepsis compel regular surveillance to enhance data-driven decision-making. Accordingly, this study aimed to assess the phenotypic epidemiology and antimicrobial resistance profiles of bacteria isolated from clinically suspected neonatal sepsis in Ethiopia. Methods A total of 342 neonates suspected of clinical sepsis were randomly included in a prospective observational study conducted at the neonatal intensive care unit (NICU) of Jimma medical center (JMC) from May 2022 to July 2023. Blood samples were collected from each neonate and subjected to a culture test for identification of bacterial isolates and their antibiotic resistance profiles following the standardized guidelines. The laboratory results, along with relevant clinical data, were recorded using WHONET and analyzed using STATA software. Results Out of the 342 blood samples that were analyzed, 138 samples (40.4%, 95% CI: 35.1–45.6, P<0.01) exhibited proven bacterial infection. The infection rates were notably higher in males with 85/138 (61.6%, 95% CI: 53.4–69.8, P<0.01) and neonates aged 0–3 days with 81/138 (58.7%, 95% CI: 50.5–66.9, P<0.01). The majority of the infections were attributed to Gram-negative bacteria, accounting for 101/138(73.2%, 95% CI: 65.6–80.7) cases, with 69/101(68.3%, 95% CI: 63.8–72.8) cases involving ESBL-producing strains, while Gram-positive bacteria were responsible for 26.8% (95% CI: 19.3–34.4) of the infections. The predominant isolates included Klebsiella pneumoniae (37.7%, 95% CI: 29.6–45.8), Coagulase-negative Staphylococci (CoNs) (20.3%, 95% CI: 13.6–27.0), and Acinetobacter species (11.6%, 95% CI: 6.0–17.1). Of the total cases, 43/72 (59.7%, 95% CI: 48.4–71.1, P<0.01) resulted in mortality, with 28/72 (38.9%, 95% CI: 27.70–50.1, P<0.03) deaths linked to Extended-Spectrum Beta-Lactamase (ESBL)-producing strains. Klebsiella pneumoniae displayed high resistance rates to trimethoprim-sulfamethoxazole (100%), ceftriaxone (100%), cefotaxime (98.1%), ceftazidime (90.4%), and gentamicin (84.6%). Acinetobacter species showed resistance to ampicillin (100%), cefotaxime (100%), trimethoprim-sulfamethoxazole (75%), ceftazidime (68.8%), chloramphenicol (68.8%), and ceftriaxone (68.8%). Likewise, CoNs displayed resistance to ampicillin (100%), penicillin (100%), cefotaxime (86.0%), gentamicin (57.2%), and oxacillin (32.2%). Multidrug resistance was observed in 88.4% (95% CI: 81.8–93.0) of isolates, with ESBL-producers significantly contributing (49.3%, 95% CI: 45.1–53.5). Furthermore, 23.0% (95% CI: 15.8–31.6) exhibited a prevalent resistance pattern to seven distinct antibiotic classes. Conclusion The prevalence and mortality rates of neonatal sepsis were significantly high at JMC, with a notable surge in antibiotic and multidrug resistance among bacterial strains isolated from infected neonates, specifically ESBL-producers. These resistant strains have a significant impact on infection rates and resistance profiles, highlighting the requisite for enhanced diagnostic and antimicrobial stewardship, stringent infection control, and further molecular characterization of isolates to enhance neonatal survival.


Introduction
Neonatal sepsis presents a major global public health issue, affecting millions of neonates worldwide each year and worsened by antimicrobial resistance [1].It consists of two distinct categories: early-onset (days of life 0-3) and late-onset (days of life 4 or later), determined by timing, transmission mode, and causative organisms [2].Bacteria are the primary culprits in categories, contributing to the fragile epidemiology of the infection, and exacerbating the rundown global crisis of antimicrobial resistance [3].
In the battle against this crisis, blood culture testing remains the gold standard for identifying bacterial causes and guiding treatment options.However, the ongoing preference for less accurate clinical diagnostic methods over blood culture testing complicates the epidemiology of neonatal sepsis [4].However, the persistent preference for less accurate clinical diagnostic approaches over blood culture testing complicates the epidemiology of neonatal sepsis [5].Consequently, the global prevalence of neonatal sepsis exhibits significant variation, with rates ranging from 25% to 60% across different neonatal populations worldwide [6,7].This variability is influenced by the diagnostic methods employed, the types of bacteria implicated, and the specific geographic locations where these cases are observed [8].For instance, in regions like Saudi Arabia and Bangladesh, neonatal sepsis commonly involves both Gram-positive and Gram-negative bacterial strains, including Enterobacter, Escherichia coli (E.coli), Klebsiella pneumoniae (K.pneumoniae), Acinetobacter baumannii, and Group B Streptococcus (GBS) [9,10].Similarly, in countries like Pakistan, Egypt, and India, prevalent pathogens contributing to neonatal sepsis include Salmonella Typhi, Coagulase-negative staphylococci (CoNs), and Staphylococcus aureus (S. aureus), respectively [11][12][13].In Jimma, Ethiopia, the primary pathogens identified in neonatal sepsis cases are Klebsiella species (36.6%),CoNS, 19.7%), and S. aureus (18.3%) [14].
Notably, the bacteria commonly encountered in healthcare settings often exhibit resistance to first-line antibiotics used in the treatment of sepsis, such as ampicillin, gentamicin, and cefoxitin.In less developed areas, a significant percentage of these bacteria demonstrate resistance, with rates ranging from 50% to 88% [8].Notably, strains of E. coli, (S. aureus), and K. pneumoniae exhibit significant resistance to antibiotics such as amoxicillin, cephalosporins, aminoglycosides, and quinolones across several countries [10].Within Ethiopian hospitals, including the current study setting, Gram-positive bacteria often show high resistance rates to penicillin (98.9%) and ceftriaxone (91.3%), while Gram-negative bacteria display resistance to ampicillin (100%), gentamicin (83.2%), and ceftriaxone (83.2%) [15].
Moreover, the rise in antimicrobial resistance, driven by various bacterial mechanisms, continues to pose a catastrophic public health threat [16].Among these threats, the emergence and dissemination of Extended-Spectrum Beta-Lactamase (ESBL) strains and the increase in multidrug-resistant (MDR) strains created an ominous future [17,18].These strains demonstrate a delicate epidemiology that varies across regions, settings, and even among different strains, with a rising global prevalence of ESBL-producing and MDR strains [17].For instance, the prevalence of ESBL-producing isolates has been reported at 42.7% in China [19], 98% in Zambia [20] and 54% in Ethiopia [21].Similarly, MDR has been reported at 64.7% in China [19], 70% in Tanzania [22] and 84% in a previous study conducted in Ethiopia [23] indicating spiraling global spread [1].
Consequently, the global partners are advocating for collective action to combating sepsis by focusing on various key areas.These include detecting the causative pathogens, monitoring trends in antimicrobial resistance, developing effective infection control measures, improving diagnostic capabilities, and identifying emerging threats worldwide.This concerted effort aims to alleviate the global threat posed by sepsis and safeguard future generations through enhanced detection and effective management [24].Despite these efforts, statistical data consistently highlights the significant role of bacterial pathogens in the onset of neonatal sepsis, compounded by the challenge of antimicrobial resistance.Concerningly, bacterial isolation and antimicrobial resistance profiling are often limited or inconsistent in many countries, including Ethiopia.Therefore, this study seeks to delve into the phenotypic epidemiology and antimicrobial resistance profiles of bacterial isolates in neonatal sepsis.

Study design, period and setting
A descriptive cohort study was executed at the Jimma Medical Center (JMC), a tertiary and referral teaching medical center in Ethiopia, from May 1, 2022 to July 31, 2023.JMC serves approximately 15 million outpatients and 16,000 inpatients annually across its various departments, which operate 24 hours a day.The pediatric department, which manages a pediatric and neonatal intensive care unit (PNICU), admits an average of 75 neonates per month for various complaints and provides routine neonatal care services.Neonates in the PNICU were diagnosed with sepsis by a pediatrician using national guidelines and suggested laboratory parameters [25].Samples for recommended laboratory tests were collected by trained nurses and transferred to the main laboratory department of the center, which is situated around 200 meters away from the PNICU.The transferred samples were checked for quality, given a specific code, and processed, with particular blood culture and antimicrobial sensitivity tests (AST) performed in a well-equipped and internationally accredited microbiology sub-unit.The individual patient records of the center, including demographics, diagnosis, treatment, and treatment outcomes, were summarized monthly and reported to the Health Information Management System office, which utilizes the District Health Information System software to store the data.

Study population, sample size estimation and sampling technique
The sampling of neonates admitted to the JMC NICU before their 28 th birthday was predated, and eligible subjects of either sex were included using a systematic random sampling approach.The study used STATCAL and the single proportion formula to determine the required sample size of 342 neonates, considering a 36% prevalence of neonatal bacteremia, a 95% CI, and a 5% margin of error [26,27].The research team employed a random selection process to identify neonates with sepsis who exhibited at least one of the clinical signs: fever (>38˚C) or hypothermia (<36˚C), rapid breathing (>60 breaths/minute), severe chest indrawing, poor feeding, seizure, lethargy, or unconsciousness, and two hematologic criteria: total leukocyte count <5000 or >12,000 cells/m3, absolute neutrophil count <1500 cells/mm3 or >7500 cells/mm3, ESR >15/h, and platelet count <150,000 or >440,000 cells/mm3)] [28].The study involved close (every hour) monitoring of selected participants within the first 6 hours of hospital admission, followed by daily monitoring throughout their inpatient stay or for up to 28 days.During the monitoring period, the study team documented the index date for each participant based on assessments and assertions made by the ward pediatrician (death, or improvement).

Inclusion criteria
The study included all neonates with suspected clinical sepsis eligible for antibiotic treatment, with documented blood culture results, and treatment outcomes.

Exclusion criteria
The study excluded neonates with incomplete records, severe congenital anomalies, preenrollment antibiotic treatment, antibiotic allergies, contaminated blood samples, and nonsepsis-related deaths.

Data source, data collection tool & procedure
The study utilized an Android-based Kobo-Collect application and a validated questionnaire to collect data from neonatal guardians, primarily mothers.The data collection process involved face-to-face interviews conducted by four BSc nurses after obtaining formal written consent from the parent or guardian of neonates.The collected data covered socio-demographics, obstetrics and delivery details, and neonatal factors.Finally, the collected data were merged with the specific laboratory data of individual neonatal participants that was uploaded into the WHONET software during the laboratory work.

Blood sample collection and pathogen profiling
A trained and experienced phlebotomist expertly extracted 1-3 ml of blood from various peripheral veins, commonly in the arm, employing strict aseptic techniques.The obtained blood samples were then introduced into Brain Heart Infusion Broth bottles using a doubleneedle method.Each sample was meticulously labeled and promptly transported to the microbiology laboratory for immediate processing.In the laboratory, the samples were cultured on Mac-Conkey Agar and blood agar plates (supply of Liofilchem1, Italy).The culture plates were incubated in an aerobic environment at 37˚C and monitored daily for seven days, with observations made within a time frame of 18-24 hours.For colonies observed on the plates, further analysis was performed for identification of the specific bacterial types.To identify Grampositive isolates, a range of methods outlined in the CLSI 2023 guidelines were employed, including Gram staining, observation of colony characteristics, and assessment of biochemical properties such as catalase, DNAse agar, Mannitol Salt Agar, and hemolysis on blood agar plates.On the other hand, Gram-negative bacilli were further identified through various biochemical tests, including triple sugar iron (TSI), lysine iron agar (LIA), motility in indole, ornithine (MIO), citrate, urease, and oxidase enzyme production.Blood culture bottles that showed bacterial growth and were consistent with the symptoms of neonatal infection were considered positive.However, if a neonate's clinical symptoms were not consistent with a positive blood culture, particularly for coagulase-negative Staphylococcus or Staphylococcus aureus, the attending pediatrician concluded that the positive result was due to contamination [29].

Phenotypic screening and confirmation of ESBLs
In this study, Gram-negative bacterial isolates were investigated, focusing on those with small inhibition zones when exposed to ceftazidime 30 μg (�22mm), Ceftriaxone 30μg(�25mm) or cefotaxime (30 μg) (�27mm).The isolates were initially identified using a combination disc screening test and confirmed with a double disc diffusion test.The tests involved placing discs containing ceftazidime 30 μg or cefotaxime 30 μg alone and ceftazidime or cefotaxime 30 μg with clavulanic acid on a bacterial culture.An increased zone of inhibition (�5mm) with the combined disc when compared to a single ceftazidime or cefotaxime disc indicated the phenotypic evidence of extended-spectrum beta-lactamase (ESBL) production.Positive and negative controls, including ESBL-producing Klebsiella pneumonia ATCC 700603 and non-ESBL-producing E. coli ATCC 25922 control strains, were used during the laboratory analysis to ensure the reliability of the results [29].

Data quality management and control
Data quality was assured through the translation of the data collection tool into local languages, specifically Afaan-Oromo and Amharic, and then back-translation to the original version.Using the translated tool, a trial run of the tool was performed on 5% of a similar population outside the study facility before the actual data collection.Subsequently, the validated and configured data tools were integrated into Kobocollect, and all data collectors underwent comprehensive two-day training.The questionnaire was loaded onto their Android devices, making data collectors well-prepared and acquainted with the tools.During the data collection process, the researcher regularly reviewed the collected and uploaded data to ensure accuracy, completeness, clarity, and consistency and to uphold the quality and dependability of the data.
Likewise, the laboratory kits, media, antibiotic discs, and other consumables were purchased from a reputable and standardized supplier.Before using the in-house-prepared culture media plates for real samples, each batch underwent sterilization checks.The laboratory analysis strictly adhered to standard operating procedures to maintain consistency and accuracy.Control strains, specifically S. aureus (ATCC-25923) for Gram-positive bacteria and P. aeruginosa (ATCC-27853) and E. coli 35218 for Gram-negative bacteria, were employed for the purpose of isolate detection and antimicrobial susceptibility testing.Before the actual data analysis, data profiling was conducted using frequency distributions and cross-tabulations.

Ethical issues
The research study obtained approval from the Jimma University Institute of Health Science Review Board on February 9, 2022, under reference number JUIRB32/2022.Following this, it was granted permission by the Jimma Medical Center Ethical Committee on February 22, 2022, with reference number THRPGn/344/2022.In adherence to the Helsinki Declaration, the researchers obtained written informed consent from the primary guardian of each neonate prior to data collection, ensuring compliance with the established protocol.

Data analysis
The data management process effectively utilized two powerful tools, STATA version 16.0 and WHONET 2022 software.WHONET, specialized software for analyzing antimicrobial resistance patterns in microbiology data, was employed to store and interpret antimicrobial resistance breakpoints and perform interim analysis of resistance profiles.Concurrently, STATA software was used to provide comprehensive descriptions of socio-demographic patterns, bacterial isolate epidemiology, and antimicrobial resistance profiles.Statistical techniques were applied to express categorical variables through absolute frequencies (N), percentages (%), confidence levels (CI), and visualizations.The results of the analyses were effectively communicated using well-designed tables, figures, and informative narrative texts.

Socio-demographic and clinical profile of participants
The study recruited a cohort of 342 neonates from rural (51.2%) and urban (48.8%) settings.Among these neonates, the majority (75.1%) were born to mothers aged 20-34 years, with males making up 60.2% of the group.A significant number (57.6%) of neonates were admitted within the initial 3 days of life.Roughly 68.1% of neonates were born at or after 36 weeks of gestation, hinting at a preterm birth rate among the rest.The vast majority (89.2%) of births occurred in healthcare facilities, with a substantial portion (74.6%) being non-cesarean deliveries compared to 25.1% delivered via cesarean section.Additionally, 72.5% of neonates had a birth weight of � 2500 grams, while 27.5% were classified as low birth weight.Early-onset sepsis was identified in 65.5% of neonates, while late-onset sepsis affected 34.5% of cases.Concerning the source of infection, 53.5% were community-acquired, while 46.5% were hospitalacquired.Upon admission, all neonates received empirical antibiotic therapy, primarily consisting of ampicillin and gentamicin (59.6%).Eventually, 78.9% of neonates showed improvement and were discharged, while 21.1% risked sepsis attributed death (Table 1).

Epidemiology of bacterial isolates in neonatal sepsis
Out of the 342 blood samples that were analyzed, 138 samples (40.4%, 95% CI: 35.1-45.6,P<0.01) exhibited proven bacterial infection.The infection rates were notably higher in males
The antibiotic resistance profile of the isolates revealed significant variability, ranging from 5.9% for meropenem to 100% for ampicillin.The highest resistance rates were observed for ampicillin at 100%, ceftriaxone at 95%, penicillin at 94.6%, cefotaxime at 93.5%, and ceftazidime at 88.1%, consistent with studies conducted in regions such as Egypt [49] and parts of Ethiopia including Bahirdar [41] and Hawasa [36].Gram-negative bacteria exhibited alarmingly high resistance to ampicillin at 100%, cefotaxime at 99.0%, ceftriaxone at 95.0%, sulfamethoxazole-trimethoprim at 93.1%, and ceftazidime at 88.1%.The resistance data revealed Klebsiella pneumoniae with the highest percentage of resistant isolates, showing resistance to sulfamethoxazole-trimethoprim at 100%, ceftriaxone at 100%, cefotaxime at 98.1%, and gentamicin at 84.6%, followed closely by Acinetobacter spp. with 100% resistance to ampicillin and cefotaxime, and 68.8% to gentamicin.Coagulase-negative Staphylococci also showed high resistance to ampicillin, penicillin G, and cefotaxime at 86%.Characteristically, the dominance of Gram-negative bacteria in the resistant profile resembles previous study results across several countries, including German [7], India [32], Tanzania [22], China [19], India [42], and Ethiopia in Addis Abeba [23].The possible reason for the dominance of Gram-negative bacteria in resistant profiles might be linked to various mechanisms of drug resistance.Specifically, Gram-negative bacteria possess an outer membrane that acts as a physical barrier, making it difficult for antibiotics to penetrate the cell wall [50].They also have efflux pumps that actively remove antibiotics from the cell, diminishing the effectiveness of these drugs, while some harbor plasmids carrying genes for resistance [51].Additionally, these bacteria produce beta-lactamase enzymes, including ESBLs, which can degrade a broader range of beta-lactam antibiotics, thereby ensuring their dominance in antimicrobial resistance [52,53].
In the context of ESBL-production, this study found that 68.3% of the bacterial isolates analyzed exhibited ESBL activity.Notably, K. pneumoniae was the most prevalent strain at 58.0%, followed by Klebsiella ozaenae at 12.0% (8 out of 69), Acinetobacter species at 7.0%, and Klebsiella oxytoca at 6.0%.The prevalence of ESBL-producing isolates in this study was higher than the rates observed in Tanzania [54] and 54.0% from a prior Ethiopia study [21].However, the isolate distribution is consistent with reports in Tanzania [22], Nepal [30], China [19], India [42], and prior result in Ethiopia [23], which also highlighted the dominance of ESBL-producing Gram-negative bacteria in neonatal sepsis.Further, it was observed that ESBL-producing strains exhibited significantly higher resistance levels to commonly prescribed antibiotics (Piperacillin-tazobactam, Amoxicillin-clavulanate, Sulfamethoxazole-trimethoprim, Ciprofloxacin, Gentamicin, Cefotaxime, Ceftazidime, and Ceftriaxone) compared to non-ESBL producers resembling the report in Pakistan [11] and Tanzania [55].The study found that ampicillin resistance was identical across all ESBL-producing and non-ESBL-producing strains, suggesting a common mechanism.However, non-ESBL-producing strains exhibited significantly higher resistance levels to chloramphenicol and meropenem antibiotics compared to ESBL-producing strains.This could be due to the presence of additional beta-lactamase enzymes beyond ESBLs, such as carbapenem-hydrolyzing Klebsiella pneumoniae carbapenemase (KPC), metallo-beta-lactamase New Delhi metallo-beta-lactamase (NDM), and variants of the oxacillinase beta-lactamase class [56].The clinical importance of ESBL-producing bacteria is highlighted in the literature, particularly due to the increasing virulence of Gram-negative pathogens in vulnerable groups like neonates.This poses a significant transmission risk in healthcare settings, compounded by factors such as hospital-acquired infections, preterm births, cesarean deliveries, and low birth weights commonly observed in population under this study [21].
Finally, the study revealed a high prevalence of multidrug resistance (MDR) in isolated bacteria, with an occurrence rate of 88.4% among all isolates.This rate is slightly higher than that in China (78.3%) [19] and a previous study in Ethiopia (84%) [23].Notably, Gram-negative strains showed a higher MDR rate at 98.0% compared to Gram-positive strains at 62.2%, consistent with earlier findings in Ethiopia [57].Moreover, Gram-negative isolates producing ESBLs had a MDR rate of 98.6%, highlighting the challenge in treating ESBL-producing bacteria.ESBL-producing Gram-negative bacteria accounted for 49.3% of MDR cases, aligning with previous study results [58].ESBL-producing isolates displayed more diverse patterns of multidrug resistance across a wider range of antibiotics compared to non-ESBL-producing Gramnegative and Gram-positive bacteria.The dominance of Gram-negative bacteria, especially with ESBL production, corresponds with findings from Tanzania [59].The resilience of MDR Gram-negative bacteria can be attributed to their complex outer membrane structure, efflux pumps, porin channels, and advanced antibiotic targets, further enhanced by ESBL enzyme production.The variations in study results are expected due to the interplay of factors favoring resistance in Gram-negative bacteria, which interact with a variety of other variables operating differently for Gram-positive bacteria.These variables likely influence the prevalence of bacterial antimicrobial resistance across geographical regions, healthcare settings, and study populations, leading to distinct results [17].
In general, bacteria epidemiology and resistance profiles are influenced by various factors, causing temporal and spatial variability.To tackle this issue, our recent study utilized larger sample sizes, an observational approach, and a certified laboratory to minimize these variables' impact.We employed randomized participant selection and a prospective design, conducting blood culture tests at a single study center for validity.By focusing on a single research center, we could control variables such as location, staff, resources, and procedures.However, it's essential to carefully consider the limitations of this approach when inferring the findings to the broader population beyond the study setting.

Conclusion and recommendation
The study has identified a high prevalence of confirmed sepsis cases and associated mortalities among neonates presenting with clinically suspected sepsis.Pathogens such as Klebsiella pneumoniae, Coagulase-negative Staphylococci, Acinetobacter species, and Klebsiella ozaenae primarily influence the epidemiology of neonatal sepsis by producing Extended-Spectrum Beta-Lactamases (ESBLs) in Klebsiella and Acinetobacter species, leading to antimicrobial resistance and multidrug resistance (MDR).These multidrug-resistant strains pose a significant challenge to current treatment approaches, necessitating the prompt development of innovative empiric antibiotic regimens.The findings of the study emphasize the critical importance for neonatal intensive care units to prioritize infection prevention strategies, reliable diagnostic stewardship, tailored antimicrobial stewardship programs, and well-informed decisions based on surveillance data.Further research is vital to elucidate the factors driving widespread antibiotic resistance, particularly in identifying resistance genes in ESBL-producing isolates, to guide the development of strategies to combat this escalating public health threat.